Project Summary/Abstract Successful treatments of refractory SLE have been reported with allogeneic but not autologous BM-derived mesenchymal stem/stromal cells (MSCs). Multiple studies have documented the immunosuppressive function of MSCs, as well as their ability to reverse disease in mouse models of SLE. Defective MSC functions have been reported in both SLE patients and lupus-prone mice, and the mechanisms underlying these defects are largely unknown. We have identified Pbx1, a transcription factor, as the susceptibility gene corresponding to the Sle1a1 locus in the NMZ2410 model of lupus. The lupus-associated allele of Pbx1 corresponds to the dominant negative (DN) Pbx1-d splice isoform. We have evidence that Pbx1-d impairs MSC functions in vitro and in vivo. B6.Sle1a1 mice differ from congenic B6 mice only by the origin of one protein-coding gene, Pbx1. Sle1a1-MSCs express Pbx1-d, and show impaired immunosuppressive abilities in vitro and in vivo, as well as a gene signature associated with inflammatory innate functions. This supports the hypothesis that Pbx1-d alters the functions of stem cells in a manner that is similar to Pbx1 deficiency. A subset of differentially expressed genes in the inflammation/innate immunity pathway is predicted to be direct Pbx1 targets. We hypothesize that the SLE-associated allele of Pbx1 impairs MSC functions, and that this impairment contributes to lupus pathogenesis, in addition to the effect that it has on T cells. Specifically, Pbx1-d impairs MSC self-renewal and immunosuppression while favoring the expression of inflammatory genes. We propose to test this hypothesis by initially focusing on the inflammatory and innate immunity genes predicted to be differentially regulated by Pbx1 with the two following specific aims: SA1. Molecular characterization of the MSC inflammatory and innate immunity pathways regulated by Pbx1-d. We will focus on a panel of 26 genes identified by RNA-Seq as differentially expressed between Sle1a1 and B6-MSCs by at least two-folds, and that contain at least one predicted Pbx1 binding site. The message expression of these genes will be validated in Sle1a1 as well as TC-MSCs as compared to B6- MSCs, and protein validation will be performed with the best suited method. The regulation of validated genes by Pbx1 will be analyzed by ChIP-qPCR. Validated Pbx1 binding sites will be further analyzed to compare transregulation between a MSC cell line (MC3T3) expressing either Pbx1-b (resistance isoform) or Pbx1-d (susceptibility isoform) by ChIP-qPCR, luciferase assays and EMSA. Finally, the expression of the validated genes as well as associated genes in their pathway will be compared between MC3T3-Pbx1-b and MC3T3- Pbx1-d. This aim will generate an extensive set of gene expression signatures induced by the expression of the two lupus-susceptibility alleles singly and in combination that will be used in SA2. SA2. Characterization of MSC functions regulated by Pbx1. We will test the functional significance of specific inflammatory and innate immunity validated genes identified in SA1 using lentiviral (LV) constructs to overexpress or knock-down (KD) genes. We will focus on two MSC functions most relevant to SLE and for which we have well-established in vitro and in vivo protocols with demonstrated efficacy: 1) immune suppression and inflammation, and 2) lineage differentiation. This aim will identify the mechanisms responsible for the MSC defects associated with Pbx1-d, as well as a means to correct them.